ACNP-MET纳米药物递送系统及其对肝癌干细胞靶向性研究
|
摘要
目的：制备特定粒径范围的活性炭纳米粒子（activated carbon nanoparticles,ACNP）与二甲双胍（metformin，MET）构建ACNP-MET纳米药物递送系统，探讨ACNP-MET对CD133~+人肝癌Huh-7细胞的靶向作用。
方法：采用球磨悬浮沉淀法制备ACNP-MET纳米药物递送系统载体ACNP；通过激光粒度分析仪、透射电镜（TEM）和原子力显微镜（AFM）对其粒径和形态进行表征；用光学显微镜（LM）和TEM观察ACNP对体外细胞形态的影响；采用乳酸脱氢酶（LDH）和组织病理学检查评价ACNP对细胞膜的影响；将一定配比的ACNP与MET超声混悬吸附，观测不同时间点未被吸附的MET的量，以吸附曲线达最高不再上升点所对应的时间作为吸附达平衡时间；将不同配比的ACNP和MET混悬液吸附达平衡后，测未被吸附的MET的量，通过等温吸附公式确定ACNP与MET的最佳配比；采用流式细胞分选技术从人肝癌细胞系Huh-7中分选出CD133~+细胞，并进行干细胞比例分析；通过对CD133~+细胞体外成球能力及增殖能力检测，评价CD133~+细胞的自我更新能力；观察在非肥胖性糖尿病/重度联合免疫缺陷小鼠(NOD/SCID)体内成瘤情况，评价CD133~+细胞的致瘤性；采用细胞增殖/毒性检测试剂盒（CCK-8）法研究ACNP-MET对体外培养CD133~+细胞的生长抑制作用；采用流式细胞术（FCM）测定ACNP-MET对Huh-7细胞中CD133~+细胞比例的影响，初步评价ACNP-MET对CD133~+细胞的靶向作用；用光学显微镜观察ACNP-MET对CD133~+细胞的成球率的影响；应用“微肿瘤”模型评价ACNP-MET对肿瘤干细胞的敏感性。
结果：球磨悬浮沉淀法制备的ACNP颗粒大小均匀、形状规则、近似球状、表面光滑，平均粒径为188nm。光镜下观察发现ACNP对细胞有很好的亲和性，细胞生长状态良好；电镜下观察,细胞轮廓明显，核膜完整，核内常染色质均匀分散，核仁大而清晰，与对照组比较未见异常。不同浓度的ACNP作用不同时间，细胞LDH漏出量与对照组相比均无显著性差异(P>0.05)。ACNP腹腔注射后3个月解剖小鼠，腹腔内未见水肿、渗出和粘连等炎症反应；病理组织学检查腹壁及腹膜组织间隙、腹膜淋巴样组织、腹腔淋巴结及淋巴样组织内有大量的ACNP，但其周围组织未见炎症细胞浸润、渗出和水肿；电镜下观察，淋巴细胞的细胞核内有ACNP，但是细胞及亚细胞结构依然正常。ACNP静脉注射后20天及6个月，观察小鼠心、肝、脾、肺和肾组织的病理切片发现，在上述组织中的ACNP周围均未见组织粘连，水肿，渗出等病理现象，脑组织中也未见ACNP。
紫外可见分光光度计全波长扫描结果显示，MET在233nm波长处有最大吸收峰，而阴性对照溶液无干扰。故选择233nm为测定波长。在1～10μg/ml范围内，MET的吸光度A与其浓度C呈直线正相关。以吸光度A对浓度C线性回归得标准曲线方程为：A=0.00479+0.08137C（r~2=0.9995，P<0.01）。在25℃时，4h左右ACNP吸附MET达到平衡状态，且随着时间的延长吸附量不再发生明显变化；室温条件下，所得等温方程式为C/X=-0.00531+0.00399C（r~2=0.995，P<0.01），ACNP对MET的饱和吸附量Xm=256.17mg/g。1g ACNP吸附256.17mg MET达到饱和。故ACNP-MET中MET与ACNP质量比为1：4。利用流式细胞术富集Huh-7细胞中CD133~+细胞，分选前的CD133~+细胞的百分比为6.08%，分选后再次测试所得细胞中CD133~+细胞的百分比为92.81%，统计学分析表明，分选前后有显著差异（P﹤0.05）。CD133~+细胞体外无血清培养3d即可成球且生长速度较CD133-细胞快。1×10~4CD133~+细胞在NOD/SCID小鼠体内21d左右即可形成异种移植瘤，测量其体积分别为57.43mm~3、57.21mm~3、52.51mm~3和53.66mm~3；光镜下观察小鼠腋下肿块病理组织切片可见，该组织有完整的肿瘤包膜，巨大的肿瘤细胞核，核仁可见，异型性明显，坏死后残存的细胞核碎片呈黑色点状，内部形成坏死区；而1×10~4CD133-细胞却无一（0/8）成瘤。在体外增殖抑制实验中，含有相同剂量的ACNP-MET组与MET相比，ACNP-MET对CD133~+及CD133-细胞均有明显增殖抑制作用，呈剂量依赖性，各组之间的统计学结果P值大都维持在0.001以下。MET及ACNP-MET对CD133-细胞的IC_(50)分别为2.514×104和316.997μg/ml；MET及ACNP-MET对CD133~+细胞的IC_(50)分别为64.509和2.847μg/ml，表明二者对CD133~+细胞的抑制率明显大于对CD133-细胞的抑制率。ACNP组对CD133~+及CD133-细胞的抑制率最大不超过6%左右；与含有相同剂量的ACNP组相比ACNP-MET与MET组对CD133~+及CD133-细胞的抑制率具有显著性差异（P<0.01）。通过FCM分析，MET对CD133~+肝癌Huh-7有抑制作用，但ACNP-MET对其作用更强，统计学分析具有显著性差异（P<0.01），呈剂量依赖性。在成瘤率实验中，200μg/ml的MET组与空白对照组和ACNP组比较，能减少CD133~+细胞所成球数；在50～200μg/ml浓度范围内, ACNP-MET对CD133~+细胞体外成球能力的抑制作用呈剂量依赖性，与MET组相比，统计学分析差异具有显著性（P<0.01）。
结论：本实验室通过球磨悬浮沉淀法制备出的ACNP与市售的相比，颗粒小且均匀，质量可控，可批量生产；活性炭有良好的生物安全性，纳米化后的ACNP依然具有良好的生物安全性；以CD133为表面标记的流式分选法，可以分选出具有强自我更新能力、增殖能力和致瘤性的肝癌Huh-7干细胞；本研究构建的ACNP-MET，MET与ACNP最佳混合吸附配比为1：4，吸附时间为4h，对MET的最大吸附量为256.17mg/g；MET作为一种用于降低血糖的“老药”可以抑制肝癌干细胞的生长和增殖；含有相同剂量MET的ACNP-MET对CD133~+及CD133-肝癌Huh-7细胞的抑制作用均好于MET；ACNP本身没有杀伤CD133~+及CD133-细胞的作用，但ACNP可以显著的增强MET对肝癌干细胞的靶向性和抑制性。ACNP与MET构建的药物递送系统对肝癌干细胞具有良好的靶向性，且ACNP-MET无需与其他化疗药连用即可增强MET对肿瘤干细胞的靶向作用。
Objective TO prepare the activated carbon nanoparticals of specific particle sizeand build nano-drug delivery system with metformin and investigate the targetingeffects of ACNP-MET to the CD133~+cells from human hepatocellular carcinomaHuh-7cells line.
Materials and Methods ACNP was prepared by ball milling and deposition indistilled water. The morphology and size of ACNP were examined through laserparticle size analyzer, transmission electron microscopy (TEM) and atomic forcemicroscopy (AFM). Huh-7cells were treated with ACNP at various concentrations fordifferent durations. The alterations in morphology of cells were observed by lightmicroscopy (LM) and TEM. The lactate dehydrogenase (LDH) leakages in the culturemedium were determined with LDH activity detection kit. LDH leakage reflects theintegrality of cell membrane. Mixed suspension of ACNP and MET was prepared andadsorption was allowed under ultrasonic condition and the concentration of MET inthe fluid was measured. The time when the amount of the MET adsorbed on ACNPreached to the highest was treated as the equilibrium time. A series of differentproportion mixture of ACNP and MET were prepared and absorbed MET wasmeasured to learn the absorption equation. The best ratio of ACNP and MET wasdetermined based on the isothermal adsorption formula. CD133~+cells were sorted byflow cytometry and the percentage of them in cultured Huh-7cells was analyzed. Theself-renewing and sphere-forming ability of CD133~+cell were observed by lightmicroscope in vitro in comparison with CD133-cells. Tumor-forming ability ofCD133~+cells were observed by xenografts of them in NOD/SCID mice. Theinhibitory effects of the ACNP-MET on the proliferation of CD133~+cells were observed by CCK-8assay. The targeting effects of the ACNP-MET on the percentageof CD133~+cells in Huh-7cells were measured by flow cytometry (FCM). Weperformed tumor-sphere assays with CD133~+cells grown in the presence or absenceof ACNP-MET. The tumor-sphere sizes were measured and the alterations inmorphology were observed under LM. TO employ mammosphere-forming efficiency(MSFE) and sphere size were used as indicators of CD133~+cells-renewal andprogenitor cell proliferation.
Results The ACNP has a similar size, a regular spherical shape and a smoothsurface. The average size is188nm. LM observation showed that ACNP canaccumulate around the cells and on the surface of cells. Neither evenly distributed inthe medium, nor aggregated and settled on the bottom of culture dish. Cells lived ingood condition. Under TEM, the outline of cell was obvious; the membrane ofnucleus was complete; the euchromatin was well distributed in nucleus; the nucleoluswas big and clear. The distribution characteristic of ACNP was advantageous forimproving the drug concentrations in the microenvironment directly contacting thecells. Abdominal cavity injection of ACNP to the mice, after3months, did not lead toedema、 adhesion and inflammatory reaction as examined by histopathologicalobservation. The experiments show that a large number of ACNP exist in lymphoidtissue and exudation and edema, inflammatory cells infiltration from the surroundingtissue were not seen when examining the abdominal wall and the peritoneum,peritoneal lymphoid tissue and lymph nodes in abdominal tissues. There was someACNP in Lymphocyte nuclei but the structure of the cell and sub-cellular structureswere normal.20days and6moths after ACNP were administrated by intravenousinjection, exudation, edema, inflammatory cellsinfiltration was not seen around theACNP in mouse heart, liver, spleen, lung and kidney, and ACNP had not been foundin brain tissue at the same time. After the Huh-7cells were treated with ACNP atvarious concentrations for24,48and72h, the LDH leakage was not higher than thatof the control group(P>0.05). MET has the maximum absorption in233nm in allwavelength scanning by ultraviolet and visible spectrophotometer. The absorption at233nm was chosen as the determine wavelength of MET. In the concentration rangeof1～10μg/ml, the absorption of MET (A) had direct proportion with theconcentration (C). The equation was A=0.00~479+0.08137C（r~2=0.9995，P<0.01）.Under25℃, ACNP adsorption for MET got to stable state at4hours later with theequal temperature equation of C/X=-0.00531+0.00399C（r~2=0.995，P<0.01）. ACNP had a maximum adsorption with a value of256.17mg MET in1g ACNP. Theoptimal ratio for adsorption was MET: ACNP=1:4. Flow cytometry analysis indicatedthat the purity of CD133~+subset cells exceed90%. CD133~+subset cells were verifiedmultipotent with the ability of forming tumor spheres within3culture days. AndCD133~+subset cells were higher proliferative in vitro and had higher tumorigeniticability in vivo than CD133-subset cells within21d. And the tumor volumes were57.43,57.20,52.51and53.66mm~3. The histopathologic slides of xenografts in mouseshowed that the tissue had capsule outsides, huge cell nucleus, and nucleolus. Themorphological characteristics of cells had significant atypia. The nuclear debris wasblack. The zone of necrosis was pink. ACNP-MET significantly inhibited theproliferation of CD133~+and CD133-cells in a dose-dependent manner, with a higherinhibition rate compared with MET. The50%inhibition concentrations (IC_(50)) of METand ACNP-MET to CD133~+cells was64.509and2.847μg/ml, respectively, while toCD133-cells was2.514×10~4and316.997μg/ml respectively. ACNP groups did nothave a greater killing effect to CD133~+and CD133-cells than control groups.Compared with MET, ACNP-MET can induced the percentage of CD133~+cells inHuh-7cells in dose dependent（P<0.01） manner. In the tumor sphere-formingexperiment, the group of200μg/ml MET can reduce the numbers of CD133~+cellsspheres compared with blank control group and ACNP group. Within50～200μg/ml,ACNP-MET had significant inhibiting effects on tumor sphere-forming indose-dependent manner（P<0.01）.
Conclusions ACNP prepared by ball milling and deposition in the distilled waterhad a smaller size, a better quality than that sold in the market and it can be producedwith a big scale. ACNP had good biosafety. CD133~+cells super marker sortingmethod can enrich CD133~+cells in high purity, and CD133~+cells sorted with CD133antibody possess the characteristics of TSC. The best proportion of MET and ACNPwas1/4and the best time was four hours for adsorption. Containing the same dose ofMET, ACNP-MET groups had a more significant inhibiting effect on CD133~+andCD133-cells than MET groups. ACNP itself could not inhibit the cells but it couldboost up the targeting and inhibiting effect of MET. ACNP-MET had better targetingeffect without combination with other chemotherapy than free MET to TSC.
方法：采用球磨悬浮沉淀法制备ACNP-MET纳米药物递送系统载体ACNP；通过激光粒度分析仪、透射电镜（TEM）和原子力显微镜（AFM）对其粒径和形态进行表征；用光学显微镜（LM）和TEM观察ACNP对体外细胞形态的影响；采用乳酸脱氢酶（LDH）和组织病理学检查评价ACNP对细胞膜的影响；将一定配比的ACNP与MET超声混悬吸附，观测不同时间点未被吸附的MET的量，以吸附曲线达最高不再上升点所对应的时间作为吸附达平衡时间；将不同配比的ACNP和MET混悬液吸附达平衡后，测未被吸附的MET的量，通过等温吸附公式确定ACNP与MET的最佳配比；采用流式细胞分选技术从人肝癌细胞系Huh-7中分选出CD133~+细胞，并进行干细胞比例分析；通过对CD133~+细胞体外成球能力及增殖能力检测，评价CD133~+细胞的自我更新能力；观察在非肥胖性糖尿病/重度联合免疫缺陷小鼠(NOD/SCID)体内成瘤情况，评价CD133~+细胞的致瘤性；采用细胞增殖/毒性检测试剂盒（CCK-8）法研究ACNP-MET对体外培养CD133~+细胞的生长抑制作用；采用流式细胞术（FCM）测定ACNP-MET对Huh-7细胞中CD133~+细胞比例的影响，初步评价ACNP-MET对CD133~+细胞的靶向作用；用光学显微镜观察ACNP-MET对CD133~+细胞的成球率的影响；应用“微肿瘤”模型评价ACNP-MET对肿瘤干细胞的敏感性。
结果：球磨悬浮沉淀法制备的ACNP颗粒大小均匀、形状规则、近似球状、表面光滑，平均粒径为188nm。光镜下观察发现ACNP对细胞有很好的亲和性，细胞生长状态良好；电镜下观察,细胞轮廓明显，核膜完整，核内常染色质均匀分散，核仁大而清晰，与对照组比较未见异常。不同浓度的ACNP作用不同时间，细胞LDH漏出量与对照组相比均无显著性差异(P>0.05)。ACNP腹腔注射后3个月解剖小鼠，腹腔内未见水肿、渗出和粘连等炎症反应；病理组织学检查腹壁及腹膜组织间隙、腹膜淋巴样组织、腹腔淋巴结及淋巴样组织内有大量的ACNP，但其周围组织未见炎症细胞浸润、渗出和水肿；电镜下观察，淋巴细胞的细胞核内有ACNP，但是细胞及亚细胞结构依然正常。ACNP静脉注射后20天及6个月，观察小鼠心、肝、脾、肺和肾组织的病理切片发现，在上述组织中的ACNP周围均未见组织粘连，水肿，渗出等病理现象，脑组织中也未见ACNP。
紫外可见分光光度计全波长扫描结果显示，MET在233nm波长处有最大吸收峰，而阴性对照溶液无干扰。故选择233nm为测定波长。在1～10μg/ml范围内，MET的吸光度A与其浓度C呈直线正相关。以吸光度A对浓度C线性回归得标准曲线方程为：A=0.00479+0.08137C（r~2=0.9995，P<0.01）。在25℃时，4h左右ACNP吸附MET达到平衡状态，且随着时间的延长吸附量不再发生明显变化；室温条件下，所得等温方程式为C/X=-0.00531+0.00399C（r~2=0.995，P<0.01），ACNP对MET的饱和吸附量Xm=256.17mg/g。1g ACNP吸附256.17mg MET达到饱和。故ACNP-MET中MET与ACNP质量比为1：4。利用流式细胞术富集Huh-7细胞中CD133~+细胞，分选前的CD133~+细胞的百分比为6.08%，分选后再次测试所得细胞中CD133~+细胞的百分比为92.81%，统计学分析表明，分选前后有显著差异（P﹤0.05）。CD133~+细胞体外无血清培养3d即可成球且生长速度较CD133-细胞快。1×10~4CD133~+细胞在NOD/SCID小鼠体内21d左右即可形成异种移植瘤，测量其体积分别为57.43mm~3、57.21mm~3、52.51mm~3和53.66mm~3；光镜下观察小鼠腋下肿块病理组织切片可见，该组织有完整的肿瘤包膜，巨大的肿瘤细胞核，核仁可见，异型性明显，坏死后残存的细胞核碎片呈黑色点状，内部形成坏死区；而1×10~4CD133-细胞却无一（0/8）成瘤。在体外增殖抑制实验中，含有相同剂量的ACNP-MET组与MET相比，ACNP-MET对CD133~+及CD133-细胞均有明显增殖抑制作用，呈剂量依赖性，各组之间的统计学结果P值大都维持在0.001以下。MET及ACNP-MET对CD133-细胞的IC_(50)分别为2.514×104和316.997μg/ml；MET及ACNP-MET对CD133~+细胞的IC_(50)分别为64.509和2.847μg/ml，表明二者对CD133~+细胞的抑制率明显大于对CD133-细胞的抑制率。ACNP组对CD133~+及CD133-细胞的抑制率最大不超过6%左右；与含有相同剂量的ACNP组相比ACNP-MET与MET组对CD133~+及CD133-细胞的抑制率具有显著性差异（P<0.01）。通过FCM分析，MET对CD133~+肝癌Huh-7有抑制作用，但ACNP-MET对其作用更强，统计学分析具有显著性差异（P<0.01），呈剂量依赖性。在成瘤率实验中，200μg/ml的MET组与空白对照组和ACNP组比较，能减少CD133~+细胞所成球数；在50～200μg/ml浓度范围内, ACNP-MET对CD133~+细胞体外成球能力的抑制作用呈剂量依赖性，与MET组相比，统计学分析差异具有显著性（P<0.01）。
结论：本实验室通过球磨悬浮沉淀法制备出的ACNP与市售的相比，颗粒小且均匀，质量可控，可批量生产；活性炭有良好的生物安全性，纳米化后的ACNP依然具有良好的生物安全性；以CD133为表面标记的流式分选法，可以分选出具有强自我更新能力、增殖能力和致瘤性的肝癌Huh-7干细胞；本研究构建的ACNP-MET，MET与ACNP最佳混合吸附配比为1：4，吸附时间为4h，对MET的最大吸附量为256.17mg/g；MET作为一种用于降低血糖的“老药”可以抑制肝癌干细胞的生长和增殖；含有相同剂量MET的ACNP-MET对CD133~+及CD133-肝癌Huh-7细胞的抑制作用均好于MET；ACNP本身没有杀伤CD133~+及CD133-细胞的作用，但ACNP可以显著的增强MET对肝癌干细胞的靶向性和抑制性。ACNP与MET构建的药物递送系统对肝癌干细胞具有良好的靶向性，且ACNP-MET无需与其他化疗药连用即可增强MET对肿瘤干细胞的靶向作用。
Objective TO prepare the activated carbon nanoparticals of specific particle sizeand build nano-drug delivery system with metformin and investigate the targetingeffects of ACNP-MET to the CD133~+cells from human hepatocellular carcinomaHuh-7cells line.
Materials and Methods ACNP was prepared by ball milling and deposition indistilled water. The morphology and size of ACNP were examined through laserparticle size analyzer, transmission electron microscopy (TEM) and atomic forcemicroscopy (AFM). Huh-7cells were treated with ACNP at various concentrations fordifferent durations. The alterations in morphology of cells were observed by lightmicroscopy (LM) and TEM. The lactate dehydrogenase (LDH) leakages in the culturemedium were determined with LDH activity detection kit. LDH leakage reflects theintegrality of cell membrane. Mixed suspension of ACNP and MET was prepared andadsorption was allowed under ultrasonic condition and the concentration of MET inthe fluid was measured. The time when the amount of the MET adsorbed on ACNPreached to the highest was treated as the equilibrium time. A series of differentproportion mixture of ACNP and MET were prepared and absorbed MET wasmeasured to learn the absorption equation. The best ratio of ACNP and MET wasdetermined based on the isothermal adsorption formula. CD133~+cells were sorted byflow cytometry and the percentage of them in cultured Huh-7cells was analyzed. Theself-renewing and sphere-forming ability of CD133~+cell were observed by lightmicroscope in vitro in comparison with CD133-cells. Tumor-forming ability ofCD133~+cells were observed by xenografts of them in NOD/SCID mice. Theinhibitory effects of the ACNP-MET on the proliferation of CD133~+cells were observed by CCK-8assay. The targeting effects of the ACNP-MET on the percentageof CD133~+cells in Huh-7cells were measured by flow cytometry (FCM). Weperformed tumor-sphere assays with CD133~+cells grown in the presence or absenceof ACNP-MET. The tumor-sphere sizes were measured and the alterations inmorphology were observed under LM. TO employ mammosphere-forming efficiency(MSFE) and sphere size were used as indicators of CD133~+cells-renewal andprogenitor cell proliferation.
Results The ACNP has a similar size, a regular spherical shape and a smoothsurface. The average size is188nm. LM observation showed that ACNP canaccumulate around the cells and on the surface of cells. Neither evenly distributed inthe medium, nor aggregated and settled on the bottom of culture dish. Cells lived ingood condition. Under TEM, the outline of cell was obvious; the membrane ofnucleus was complete; the euchromatin was well distributed in nucleus; the nucleoluswas big and clear. The distribution characteristic of ACNP was advantageous forimproving the drug concentrations in the microenvironment directly contacting thecells. Abdominal cavity injection of ACNP to the mice, after3months, did not lead toedema、 adhesion and inflammatory reaction as examined by histopathologicalobservation. The experiments show that a large number of ACNP exist in lymphoidtissue and exudation and edema, inflammatory cells infiltration from the surroundingtissue were not seen when examining the abdominal wall and the peritoneum,peritoneal lymphoid tissue and lymph nodes in abdominal tissues. There was someACNP in Lymphocyte nuclei but the structure of the cell and sub-cellular structureswere normal.20days and6moths after ACNP were administrated by intravenousinjection, exudation, edema, inflammatory cellsinfiltration was not seen around theACNP in mouse heart, liver, spleen, lung and kidney, and ACNP had not been foundin brain tissue at the same time. After the Huh-7cells were treated with ACNP atvarious concentrations for24,48and72h, the LDH leakage was not higher than thatof the control group(P>0.05). MET has the maximum absorption in233nm in allwavelength scanning by ultraviolet and visible spectrophotometer. The absorption at233nm was chosen as the determine wavelength of MET. In the concentration rangeof1～10μg/ml, the absorption of MET (A) had direct proportion with theconcentration (C). The equation was A=0.00~479+0.08137C（r~2=0.9995，P<0.01）.Under25℃, ACNP adsorption for MET got to stable state at4hours later with theequal temperature equation of C/X=-0.00531+0.00399C（r~2=0.995，P<0.01）. ACNP had a maximum adsorption with a value of256.17mg MET in1g ACNP. Theoptimal ratio for adsorption was MET: ACNP=1:4. Flow cytometry analysis indicatedthat the purity of CD133~+subset cells exceed90%. CD133~+subset cells were verifiedmultipotent with the ability of forming tumor spheres within3culture days. AndCD133~+subset cells were higher proliferative in vitro and had higher tumorigeniticability in vivo than CD133-subset cells within21d. And the tumor volumes were57.43,57.20,52.51and53.66mm~3. The histopathologic slides of xenografts in mouseshowed that the tissue had capsule outsides, huge cell nucleus, and nucleolus. Themorphological characteristics of cells had significant atypia. The nuclear debris wasblack. The zone of necrosis was pink. ACNP-MET significantly inhibited theproliferation of CD133~+and CD133-cells in a dose-dependent manner, with a higherinhibition rate compared with MET. The50%inhibition concentrations (IC_(50)) of METand ACNP-MET to CD133~+cells was64.509and2.847μg/ml, respectively, while toCD133-cells was2.514×10~4and316.997μg/ml respectively. ACNP groups did nothave a greater killing effect to CD133~+and CD133-cells than control groups.Compared with MET, ACNP-MET can induced the percentage of CD133~+cells inHuh-7cells in dose dependent（P<0.01） manner. In the tumor sphere-formingexperiment, the group of200μg/ml MET can reduce the numbers of CD133~+cellsspheres compared with blank control group and ACNP group. Within50～200μg/ml,ACNP-MET had significant inhibiting effects on tumor sphere-forming indose-dependent manner（P<0.01）.
Conclusions ACNP prepared by ball milling and deposition in the distilled waterhad a smaller size, a better quality than that sold in the market and it can be producedwith a big scale. ACNP had good biosafety. CD133~+cells super marker sortingmethod can enrich CD133~+cells in high purity, and CD133~+cells sorted with CD133antibody possess the characteristics of TSC. The best proportion of MET and ACNPwas1/4and the best time was four hours for adsorption. Containing the same dose ofMET, ACNP-MET groups had a more significant inhibiting effect on CD133~+andCD133-cells than MET groups. ACNP itself could not inhibit the cells but it couldboost up the targeting and inhibiting effect of MET. ACNP-MET had better targetingeffect without combination with other chemotherapy than free MET to TSC.
引文
[1] AL-HAJJM, CLARKEMF. Self-renewaland solid tumor stem cells[J].Oncogene,2004,23(43):7274-728.
[2] Fillmore CM, Kuperwasser C. Human breast cancer cell lines contain stemlike cells that self-renew, give rise to phenotypically diverse progeny and survivechemotherapy[J]. Breast Cancer Res,2008,10(2): R25.
[3] Hermann PC, Huber SL, Herrler T, et al. Distinct populations of cancer stemcells determine tumor growth and metastatic activity in human pancreatic cancer[J].Cell Stem Cell2007,1(3):313-323.
[4] ChangXiao Liu. Research and application of targeting nao-drugs[J]. NanoBiomed. Eng.2011,3(2):73-83.季平.肿瘤干细胞研究进展及展望[J].重庆医学,2007,36(4)：289-290.
[5] MAEDAH, W U J, SAWAT, et al Tumor vascular permeability and the EPReffect in macromolecular therapeutics: a review[J]. J Control Release,2000,65(2):271-284.
[6] A.K. Iyer, G. Kha led, J. Fang, H. Maeda, Exploiting the enhancedpermeability and retention effect for tumor targeting[J]. Drug Discovery Today11(2006):812–818.
[7] T. Lammers, et al., Drug targeting to tumors: Principles, pitfalls and (pre-)clinical progress[J]. J. Control. Release,2012, doi:10.1016/j.jconrel.2011.09.063\SHEKHARC.
[8] Lean and mean: nanoparticlebased delivery improves performance of cancerdrugs[J]. Chem Biol,2009,16(4):349-350.
[9]曲秋莲，张英鸽，杨留中，等.纳米活性炭吸附丝裂霉素C腹腔化疗的实验研究[J].中华肿瘤杂志,2006,28(4):257-260.
[10]孙岚,张英鸽,杨留中.纳米活性炭在小鼠体内的分布、存留及淋巴靶向性[J].军事医学科学院院刊,2005,29(4):349-351.
[11]李章芳，沈洁。二甲双胍对肿瘤抑制作用的研究进展[J].中华临床医师杂志,2011,5（17）:5081-5083.
[12] Vazquez-Martin A, Oliveras-Ferraros C, Cu fi S, Del Barco S, Martin-Castillo B, Menendez JA. Metformin regulates breast cancer stem cell ontogeny bytranscrip-tional regulation of the epithelial–mesenchymal transition (EMT) status[J].Cell Cycle2010,9(18):3807-3814.
[13] Hirsch HA, Iliopoulos D, Tsichlis PN, Struhl K. Metformin selectivelytargets cancer stem cells, and acts together with chemotherapy to block tumor growthand pro-long remission[J]. Cancer Res2009,69(19):7507-7511.
[14] Shank JJ, Yang K，Ghannam J, et al., Metformin targets ovarian cancer stemcells in vitro and in vivo[J]. Gynecol Oncol (2012),http://dx.doi.org/10.1016/j.ygyno.2012.07.115.
[15] Iliopoulos D, Hirsch HA, Struhl K. Metformin decreases the dose ofchemotherapy for prolonging tumor remission in mouse xenografts involving multiplecancer cell types[J]. Cancer Res,2011,71(9):3196-201.
[1]曲秋莲，张英鸽，杨留中，等.纳米活性炭吸附丝裂霉素C腹腔化疗的实验研究[J].中华肿瘤杂志，2006，28(4):257-260.
[2]孙岚,张英鸽,杨留中.纳米活性炭在小鼠体内的分布、存留及淋巴靶向性[J].军事医学科学院院刊，2005，29(4):349-351.
[3]张李，梁寒.纳米活性炭的特性及其在胃癌治疗中的应用[J].国际肿瘤学杂志，2007，34（1）：52-55.
[4] Hagiwara A, Torii T, Sawai K, et al. Local injection of anti-cancer drugs bound tocarbon particles for early gastric cancer-a pilot study[J]. Hepatogastroenterology，2000，47(32):575-578.
[5]张英鸽,孙岚,曲秋莲.纳米活性炭的制备及应用，中国专利. ZL03131497.X.
[6]苏伟，周理.高比表面积活性炭制备技术的研究进展[J].化学工程，2005，33(2)：44-47.
[7]孙明磊，温玉明，王昌美，等.淋巴靶向平阳霉素—活性炭纳米微粒的研制[J].华西口腔医学杂志，2004，22(3)：183-185.
[8]董怡民，沓小容，姚崇顺，等.纳米活性炭对氟尿嘧啶的吸附和缓释作用的实验研究[J].中国医科大学学报，2004，33(3)：205-211.
[9]张英鸽，纳米毒理学，中国协和医科大学出版社，2010，第一版：264-265.
[10]李晓林，张艳芳，唐凯，等.纳米二氧化钛颗粒体外对人肺腺癌A549细胞的毒性效应[J].第二军医大学学报，2011，32（10）：1091-1095.
[11]吴秋云，唐萌，谢彦昕，等.不同粒径纳米二氧化硅的体外细胞膜毒性作用[J].中国生物医学工程学报，2010，29（3）：437-445.
[12]杨红，吴秋云，唐萌，等.纳米氧化硅的体外细胞毒性[J].东南大学学报(自然科学版)，2008，38（6）：1054-1060.镍对体外培养内皮细胞的影响[J].现代生物医学进展，2010，10（2）：224-228.
[1]李章芳，沈洁。二甲双胍对肿瘤抑制作用的研究进展[J].中华临床医师杂志，2011，5（17:5081-5083.
[2] Vazquez-Martin A, Oliveras-Ferraros C, Cufi S, Del Barco S, Martin-Castillo B,Menendez JA. Metformin regulates breast cancer stem cell ontogeny bytranscriptional regulation of the epithelial-mesenchymal transition (EMT) status[J].Cell Cycle2010，9:3807-14.
[3] Vazquez-Martin A, Oliveras-Ferraros C, Cu fi S, Del Barco S, Martin-Ca stillo B,Menendez JA. Metformin regulates breast cancer stem cell ontogeny bytranscrip-tional regulation of the epithelial–mesenchymal transition (EMT) status[J].Cell Cycle2010，9:3807-3814.
[4] Hirsch HA, Iliopoulos D, Tsichlis PN, Struhl K. Metformin selectively targetscancer stem cells，and acts together with chemotherapy to block tumor growth andpro-long remission[J]. Cancer Res2009，69：7507-11.
[5] Iliopoulos D, Hirsch HA, Struhl K. Metformin decreases the dose ofchemotherapy for prolonging tumor remission in mouse xenografts involving multiplecancer cell types[J]. Cancer Res2011，71:3196-201.
[6]国家药典委员会编1中华人民共和国药典(二部)[S].2005年版.北京:化学工业出版社，2005：457、附录36.
[7] Aginara A, Takahashi T, AanaiK, et al. Selective drug delivery to peritum oralregion lymphatics by local injection of aclarubicin adsorbed on activated carbonparticles in patients with breast cancer pilot study[J]. Anticancer Drug，1997，8(7):666-670.
[8] Wang Guijuan, Cao Mingli, Yu Yongfu. Study on preparation of Al-pillaredMontmorillonite and its Adsorption[J]. Industrial Minerals and Processing，2003，11:9-11.
[9] Yoshizawa T, Yamashita A, Luo Y. Fumonisin occurrence in corn from high andlow risk areas for human esophageal cancer in China [J]. Appl Environ Microbiol,1994，60(5):1626-1629.
[10] Yang Zhong, Ma Shuzheng and Zhang Yingge. Using activated carbonnanoparticles to decrease the genotoxicity and teratogenicity of anticancertherapeutic agents[J]. J Nanosci Nanotechnol.2010，10:1-7.
[11] SINGHR, Jr LILLARD JW. Nanoparticle-based targeted drug delivery[J]. ExpMol Pathol2009,86(3):215-223.
[12] TA H T, DASS C R, DUNSTAN D E. Injectable chitosan hydrogels for localisedcancer therapy[J]. J Control Release，2008，126(3):205-216.
[13] TRAN M A, WATTS R J, ROBERTSON G P. Use of liposomes as drug deliveryvehicles for treatment of melanoma[J]. Pigm Cell Melanoma R，2009，22(4):388-399.
[14] JABR-MILANE L S, van VLERKEN L E, YADAV S, et al. Multi-functionalnanocarriers to overcome tumor drug resistance [J]. Cancer Treat Rev，2008，34(7):592-602.
[15] Yih TC, Al-Fandi M. Engineered nanoparticles as precise drug delivery systems.J Cell Biochem2006，97:1184-90.
[16] Suphiya Parveen, MS, Ranjita Misra, MS, Sanjeeb K. Sahoo, PhD. Nanoparticles:a boon to drug delivery, therapeutics, diagnostics and imaging[J].Nanomedicine:Nanotechnology, Biology, and Medicine8(2012):147–166.
[1] Gupta PB, Onder TT, Jiang G, et al. Identification of selective inhibitors of cancerstem cells by high-throughput screening. Cell,138:645–659.
[2]Sharon R. Pinea, Blair Marshallb et al. Lung cancer stem cells. Disease Markers24:257–266.
[3]Yin, S., Li, J., Hu, C. et al. CD133positive hepatocellular carcinoma cells possesshigh capacity for tumorigenicity[J]. Int J Cancer.2007,120(7):1444-1450.
[4] Ma, S., Chan, K. W., Hu, L. et al. Identification and characterization oftumorigenic liver cancer stem/progenitor cells[J]. Gastroenterology.2007,132(7):2542-2556.
[5] Yang, W., Yan, H. X., Chen, L. et al. Wnt/beta-catenin signaling contributes toactivation of normal and tumorigenic liver progenitor cells[J]. Cancer Res.2008,68(11):4287-4295.
[6] Yamashita, T., Ji, J., Budhu, A. et al. EpCAM-positive hepatocellular carcinomacells are tumor-initiating cells with stem/progenitor cell features[J]. Gastroenterology.2009,136(3):1012-1024
[7] Wu, A., Wiesner, S., Xiao, J. et al. Expression of MHC I and NK ligands onhuman CD133+glioma cells: possible targets of immunotherapy[J]. J Neurooncol.2007,83(2):121-131
[8] Song, W., Li, H., Tao, K. et al. Expression and clinical significance of the stem cellmarker CD133in hepatocellular carcinoma[J]. Int J Clin Pract.2008,62(8):1212-1218
[9]Horst, D., Scheel, S. K., Liebmann, S. et al. The cancer stem cell marker CD133has high prognostic impact but unknown functional relevance for the metastasis ofhuman colon cancer[J]. J Pathol.2009,219(4):427-434
[10]Yang L, Parkin DM, Li L, et al. Time trends in cancer mortality in China:1987－1999[J]. Int J Cancer,2003,106(5):771－783.
[11] de Villa V, Lo CM. Liver transplantation for hepatocellular carcinoma inAsia[J]. Oncologist,2007,12(11):1321－1331.
[12]孙力超，赵璇，孙立新，等，肝癌干细胞抗体靶向治疗的实验，肿瘤防治研究，2011，38（6）：609-614
[13] Yang ZF, Ho DW, Ng MN, et al. Significance of CD90+cancer stem cells inhuman liver cancer[J]. Cancer Cell,2008,13(2):153－166.
[14] Al－Hajj M, Wicha MS, BenitoHernandez A, et al. Prospective identificationof tumorigenic breast cancer cells[J]. Proc Natl Acad Sci USA,2003,100(7):3983－3988.
[15]Haraguchi N, Ohkuma M, Sakashita H, et al. CD133+CD44+populationefficiently enriches colon cancer initiating cells[J]. Ann Surg Oncol,2008,15(10):2927－2933.
[16]Tirino V, Camerlingo R, Franco R, et al. The role of CD133in theidentification and characterisation of tumourinitiating cells in nonsmallcell lungcancer [J]. Eur J Cardiothorac Surg,2009,36(3):446－453.
[17]Taylor RA, Toivanen R, Risbridger GP. Stem cells in prostate cancer: treatingthe root of the problem[J]. Endocr Relat Cancer,2010,17(4): R273－R285.
[18] Ma S, Chan KW, Hu L, et al. Identification and characterization oftumorigenic liver cancer stem/progenitor cells[J]. Gastroenterology,2007,132(7):2542－2556.
[19]梁晨,单娟娟,沈俊杰.肝癌细胞系Huh7中肿瘤干细胞分离及鉴定,浙江理工大学学报,2001,28(4):602-605.
[20]Dontu G, Abdallah WM, Foley JM, et al. In vitro propagation andtranscriptional profiling of human mammary stem/progenitor cells[J]. Genes Dev,2003,17(10):1253－1270.
[21]Toma J G, Akhavan M, Fernandes KJ, et al. Isolation of multipotent adultstem cells from the dermis of mammalian skin[J]. Nat Cell Biol,2001,3(9):778－784.
[22]Yin AH, Miraglia S, Zanjani ED, et al AC133a novel marker for humanhematopoietic stem and progenitor cells[J]. Blood,1997,90:5002-5012
[23]Singh SK, Clarke D. Identification of a cancer stem cell in human braintumors[J]. Cancer Res,2003,63:58212-58218.
[24]Hadnagy A，Gaboury L，Beanlieu R，et al. SP an alysis may be used to identifycancer stem cell populations. Exp Cell Res2006;312(19):3701-3710.
[25] Ma S，Lee TK，Zheng B-J，et al. CD133+HCC cancer stem cells conferchemoresistance by preferential expression of the Akt/PKB survival pathway.Oncogene2008;27(12):1749-1758.
[1] AL-HAJJM, CLARKEMF. Self-renewal and solid tumor stem cells[J]. Oncogene,2004,23(43):7274-728.
[2] Hadnagy A，Gaboury L，Beanlieu R，et al. SP an alysis may be used to identifycancer stem cell populations[J]. Exp Cell Res2006,312(19):3701-3710.
[3] Ma S，Lee TK，Zheng B-J，et al. CD133+HCC cancer stem cells conferchemoresistance by preferential expression of the Akt/PKB survival pathway[J].Oncogene2008,27(12):1749-1758.
[4] Hirsch HA, Iliopoulos D, Tsichlis PN, et al. Metformin selectively targets cancerstem cells, and acts together with chemotherapy to block tumor growth and prolongremission[J]. Cancer Res,2009,69:7507–7511.
[5] Shank JJ, Yang K, Ghannam J,et al., Metformin targets ovarian cancer stem cellsin vitro and in vivo[J]. Gynecol Oncol,2012, DOI/10.1016/j.ygyno.2012.07.115.
[6] Alejandro V M, Cristina OF,Sonia D B, et al. The anti-diabetic drug metforminsuppresses self-renewal and proliferation of trastuzumab-resistant tumor-initiatingbreast cancer stem cells[J]. Breast Cancer Res Treat,2011,126:355–364.DOI10.1007/s10549-010-0924-x.
[7] ChangXiao Liu. Research and application of targeting nano-drugs[J]. NanoBiomed.Eng,2011,3(2),73-83.
[8] Grimshaw MJ, Cooper L, Papazisis K, et al. Mammosphere culture of metastaticbreast cancer cells enriches for tumorigenic breast cancer cells[J]. Breast Cancer Res,2008,10: R52.
[9] MAEDAH,WUJ, SAWAT, et al.Tumor vascular permeability and the EPR effectin macromolecular therapeutics: a review[J]. J Control Release,2000,65(2):271-284.
[10] A.K. Iyer, G. Kha led, J. Fang, H. Maeda, Exploiting the enhanced permeabilityand retention effect for tumor targeting, Drug Discovery Today11(2006)812–818.
[11] Dontu G, Abdallah WM, Foley JM, et al. In vitro propagation and transcriptionalprofiling of human mammary stem/progenitor cells[J]. Genes Dev2003,17:1253–1270.
[12] Dontu G, Jackson KW, McNicholas E, et al. Role of Notch signaling in cell-fatedetermination of human mammary stem/progenitor cells[J]. Breast Cancer Res,2004,6:605–615.
[13] Marquardt J U, Factor V M, Thorgeirsson S S. Epigenetic regulation of cancerstem cells in liver cancer: Current concepts and clinical implications[J]. Journal ofHepatology,2010,53:568–577.
[14] Li C, Liu VW, Chan DW, Yao KM, Ngan HY. LY294002and metformincooperatively enhance the inhibition of growth and the induction of apoptosis ofovarian cancer cells[J]. Int J Gynecol Cancer2012,22:15-22.
[1] J.D. Byrne, T. Betancourt, L. Brannon-Peppas, Active targeting schemes fornanopartic le systems in cancer therapeutics[J]. Adv. Drug Deliv. Rev.60(2008):1615–1626.
[2] Y.H. Bae, Drug targeting and tumor heterog eneity[J]. J. Control. Release133(2009):2–3.
[3] S.H. Jang, M.G. Wientjes, D. Lu, J.L.S. Au, Drug delivery and transport to solidtumors, Pharmaceutical Research[J].2003(20):1337–1350.
[4] J.R. Less, T.C. Skalak, E.M. Sevick,R.K.Jain, Microvascular architecture in amammary carcinoma: br-anching patterns and vessel dimensions[J]. Cancer Research1991(51):265–273.
[5] F. Yuan, M. Dellian, D. Fukum ura, M. Leunig, D.A. Berk, V.P. Torchilin, R.K.Jain,Vascular-permeability in a human tumor xenograft—molecular-size dependenceand cutoff size[J]. Cancer Research55(1995):3752–3756.
[6] W.G. Roberts, G.E. Palade, Neovasculature induced by vascular endothelial growthfactor is fenestrated[J]. Cancer Research57(1997):765–772.
[7] Yinghuan Li, Jie Wang, M. Guillaume Wientjes, Jessie L.-S. Au. Delivery ofnanomedicines to extracellular and intracellular compartments of a solid tumor[J].Advanced Drug Delivery Reviews64(2012):29–39.
[8]S.H.Jang, M.G.Wientjes, D.Lu,J.L.S. Au, Drug delivery and transport to solidtumors[J]. Pharmaceutical Research20(2003):1337–1350.
[9] W. Peters, M. Teixeira, M. Intaglietta, J.F. Gross, Microcirculatory studies in ratmammary carcin oma. I. Transparent chamber method, development ofmicrovasculature, and pressures in tumor vessels[J]. J. Natl. Cancer Inst.65(1980):631–642.
[10] F. Yuan, M. Dellian, D. Fukum ura, M. Leunig, D.A. Berk, V.P. Torchilin, R.K.Jain,Vascular-pe rmeability in a human tumor xenograft—molecular-size dependenceand cutoff size[J]. Cancer Research55(1995):3752–3756.
[11] C.H. Heldin, K. Rubin, K. Pietras, A. Ostman, High interstitial fluid pressure—an obstacle in cancer therapy[J]. Nat. Rev. Cancer4(2004):806–813.
[12] AL-HAJJM, CLARKEMF. Self-renewal and solid tumor stem cells[J]. Oncogene,2004,23(43):7274-728.
[13] Gupta PB, Onder TT, Jiang G, et al. Identification of selective inhibitors ofcancer stem cells by high-throughput screening[J]. Cell2009:138:645–659.
[14] Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer and cancerstem cells[J]. Nature2001，414:105–111.
[15] Jordan CT, Guzman ML, Noble M. Cancer stem cells[J]. N Engl J Med2006，355:1253–1261.
[16] Sharon R. Pinea, Blair Marshallb and Lyuba Varticovski. Lung cancer stemcells[J]. Disease Markers24(2008)257–266.
[17] Nguyen DX, Bos PD, Massague J. Metastasis: from dissemination toorgan-specific colonization[J]. Nat Rev Cancer2009;9:274–284.
[18] Kim MY, Oskarsson T, Acharyya S, Nguyen DX, Zhang XH, Norton L,et al.Tumor self-seeding by circulating cancer cells[J]. Cell2009;139:1315–1326.
[19] Jordan CT, Guzman ML, Noble M. Cancer stem cells[J]. N Engl J Med2006,355:1253–1261.
[20] Li L, Neaves WB. Normal stem cells and cancer stem cells: the niche matters[J].Cancer Res2006,66:4553–4557.
[21] Bissell MJ, Labarge MA. Context, tissue plasticity, and cancer: are tumor stemcells also regulated by the microenvironment?[J]. Cancer Cell2005,7:17–23.
[22] Polyak K, Weinberg RA. Transitions between epithelial and mesenchymal states:acquisition of malignant and stem cell traits[J]. Nat Rev Cancer2009,9:265–273.
[23] Jaiswal S, Jamieson CH, Pang WW, Park CY, Chao MP, Majeti R, et al. CD47isupregulated on circulating hematopoietic stem cells and leukemia cells to avoidphagocytosis[J]. Cell2009,138:271–285.
[24] Lu J, Fan T, Zhao Q, Zeng W, Zaslavsky E, Chen JJ, et al. Isolation of circulatingepithelial and tumor progenitor cells with an invasive phenotype from breast cancerpatients[J]. Int J Cancer2010,126:669–683.
[25] Ahn JB, Rha SY, Shin SJ, Jeung HC, Kim TS, Zhang X, et al. Circulatingendothelial progenitor cells (EPC) for tumor vasculogenesis in gastric cancerpatients[J]. Cancer Lett2009,288:124–132.
[26] Hermann PC, Huber SL, Herrler T, Aicher A, Ellwart JW, Guba M, et al.Distinctpopulations of cancer stem cells determine tumor growth and metastatic activity inhuman pancreatic cancer[J]. Cell Stem Cell2007,1:313–323.
[27] Aktas B, Tewes M, Fehm T, Hauch S, Kimmig R, Kasimir-Bauer S. Stem celland epithelial-mesenchymal transition markers are frequently overexpres-sed incirculating tumor cells of metastatic breast cancer patients[J]. Breast Cancer Res2009;11:R46.
[28] Liu R, Wang X, Chen GY, Dalerba P, Gurney A, Hoey T, et al. The prognosticrole of a gene signature from tumorigenic breast-cancer cells[J]. N Engl J Med2007;356:217–226.
[29] Yang ZF, Ngai P, Ho DW, Yu WC, Ng MN, Lau CK, et al. Identification of localand circulating cancer stem cells in human liver cancer[J].Hepatology2008,47:919–928.
[30] Clarke MF, Fuller M. Stem cells and cancer: two faces of eve[J]. Cell2006;124:1111–1115.
[31] Huff CA, Matsui W, Smith BD, Jones RJ. The paradox of response and survivalin cancer therapeutics. Blood2006;107(2):431-434.
[32] SINGHR, Jr LILLARD JW. Nanoparticle-based targeted drug delivery[J]. ExpMol Pathol2009,86(3):215-223.
[33] TA H T, DASS C R, DUNSTAN D E. Injectable chitosan hydrogels for localisedcancer therapy[J]. J Control Release,2008,126(3):205-216.
[34] TRAN M A, WATTS R J, ROBERTSON G P. Use of liposomes as drug deliveryvehicles for treatment of melanoma[J]. Pigm Cell Melanoma R,2009,22(4):388-399.
[35] JABR-MILANE L S, van VLERKEN L E, YADAV S, et al. Multi-functionalnanocarriers to overcome tumor drug resistance [J]. Cancer Treat Rev,2008,34(7):592-602.
[36] Yih TC, Al-Fandi M. Engineered nanoparticles as precise drug deliverysystems[J]. J Cell Biochem2006;97:1184-90.
[37] Suphiya Parveen, MS, Ranjita Misra, MS, Sanjeeb K. Sahoo, PhD. Nanoparticles:a boon to drug delivery, therapeutics, diagnostics and imaging[J].Nanomedicine:Nanotechnology, Biology, and Medicine8(2012):147–166.
[38] LEEES, KIMD, YOUNY S, et al. A novel virus-mimetic nanogelvehicle[J].Angew Chem Int Ed,2008,47(13):2418-2421.
[39] LEEES, GAOZ, KIMD, et al. Super pH-sensitivemultifunctional polymericmicelle for tumor pH(e) specific TAT exposure and multidrug resistance: in vivoefficacy[J]. J ControlRelease,2008,129(3):228-236.
[40] KOBAYASHI T, ISHIDAT, OKADAY, et al. Effect of transferrinreceptor-targeted liposomal doxorubicin in P-glycoproteinmediated drug resistanttumor cells[J]. Int J Pharm,2007,329(1):94-102.
[41] MOHAJERG, LEEES, BAEYH. Enhanced intercellular retention activity ofnovel pH-sensitive polymeric micelles in wild and multidrug resistant MCF-7cells[J].PharmRes,2007,24(9):1618-1627.
[42] LEEES, NAK, BAEYH. Doxorubicin loaded pHsensitive polymeric micelles forreversal of resistant MCF-7tumor[J].J ControlRelease,2005,103(2):405-418.
[43] MAEDAH,WUJ, SAWAT, et al Tumor vascular permeability and the EPR effectin macromolecular therapeutics: a review[J]. J Control Release,2000,65(2):271-284.
[44] A.K. Iyer, G. Kha led, J. Fang, H. Maeda, Exploiting the enhanced permeabilityand retention effect for tumor targeting[J]. Drug Discovery Today11(2006)812–818.
[45] SHEKHARC. Lean and mean: nanoparticlebased delivery improves performanceof cancer drugs[J]. Chem Biol,2009,16(4):349-350.
[46] LI SD, HUANGL. Pharmacokinetics and biodistribution of nanoparticles[J].MolPharmaceutics,2008,5(4):496-504.
[47] OGAWARAK, UNK, TANAKAK, et al.In vivo antitumor effect of PEGliposomal doxorubicin(DOX) in DOX-resistant tumor-bearing mice:involvement ofcytotoxic effect on vascular endothelial cells[J]. J Control Release,2009,133(1):4-10
[48] SENGUPTA S, EAVARONED, CAPILA I, et al. Temporal targeting of tumourcells and neovasculature with a nanoscale delivery system[J].Nature,2005,436(7050):568-572.
[49] Lim G. Pelosi, M. Barisella, F. Pasini, M.E. Leon, G. Veronesi, L. Spaggiari, F.Fraggetta, A. Iannucci, M. Masullo, A. Sonzogni, F. Maffini and G. Viale, CD117immunoreactivity in stage I adenocarcinoma and squamous cell carcinoma of the lung:relevance to prognosis in a subset of adenocarcinomapatients [J]. Mod Pathol17(2004),711–721.
[50] M. Gutova, J. Najbauer, A. Gevorgyan, M.Z. Metz, Y.Weng, C.C. Shih and K.S.Aboody, Identification of uPAR-positive Chemoresistant Cells in Small Cell LungCancer[J]. PLoS ONE2(2007), e243.
[51] Zhang L, Yao HJ, Yu Y, Zhang Y, Li RJ, Ju RJ, et al. Mitochondrial targetingliposomes incorporating daunorubicin and quinacrine for treatment of relapsed breastcancer arising from cancer stem cells[J]. Biomaterials2012,33:565-582.
[2] Fillmore CM, Kuperwasser C. Human breast cancer cell lines contain stemlike cells that self-renew, give rise to phenotypically diverse progeny and survivechemotherapy[J]. Breast Cancer Res,2008,10(2): R25.
[3] Hermann PC, Huber SL, Herrler T, et al. Distinct populations of cancer stemcells determine tumor growth and metastatic activity in human pancreatic cancer[J].Cell Stem Cell2007,1(3):313-323.
[4] ChangXiao Liu. Research and application of targeting nao-drugs[J]. NanoBiomed. Eng.2011,3(2):73-83.季平.肿瘤干细胞研究进展及展望[J].重庆医学,2007,36(4)：289-290.
[5] MAEDAH, W U J, SAWAT, et al Tumor vascular permeability and the EPReffect in macromolecular therapeutics: a review[J]. J Control Release,2000,65(2):271-284.
[6] A.K. Iyer, G. Kha led, J. Fang, H. Maeda, Exploiting the enhancedpermeability and retention effect for tumor targeting[J]. Drug Discovery Today11(2006):812–818.
[7] T. Lammers, et al., Drug targeting to tumors: Principles, pitfalls and (pre-)clinical progress[J]. J. Control. Release,2012, doi:10.1016/j.jconrel.2011.09.063\SHEKHARC.
[8] Lean and mean: nanoparticlebased delivery improves performance of cancerdrugs[J]. Chem Biol,2009,16(4):349-350.
[9]曲秋莲，张英鸽，杨留中，等.纳米活性炭吸附丝裂霉素C腹腔化疗的实验研究[J].中华肿瘤杂志,2006,28(4):257-260.
[10]孙岚,张英鸽,杨留中.纳米活性炭在小鼠体内的分布、存留及淋巴靶向性[J].军事医学科学院院刊,2005,29(4):349-351.
[11]李章芳，沈洁。二甲双胍对肿瘤抑制作用的研究进展[J].中华临床医师杂志,2011,5（17）:5081-5083.
[12] Vazquez-Martin A, Oliveras-Ferraros C, Cu fi S, Del Barco S, Martin-Castillo B, Menendez JA. Metformin regulates breast cancer stem cell ontogeny bytranscrip-tional regulation of the epithelial–mesenchymal transition (EMT) status[J].Cell Cycle2010,9(18):3807-3814.
[13] Hirsch HA, Iliopoulos D, Tsichlis PN, Struhl K. Metformin selectivelytargets cancer stem cells, and acts together with chemotherapy to block tumor growthand pro-long remission[J]. Cancer Res2009,69(19):7507-7511.
[14] Shank JJ, Yang K，Ghannam J, et al., Metformin targets ovarian cancer stemcells in vitro and in vivo[J]. Gynecol Oncol (2012),http://dx.doi.org/10.1016/j.ygyno.2012.07.115.
[15] Iliopoulos D, Hirsch HA, Struhl K. Metformin decreases the dose ofchemotherapy for prolonging tumor remission in mouse xenografts involving multiplecancer cell types[J]. Cancer Res,2011,71(9):3196-201.
[1]曲秋莲，张英鸽，杨留中，等.纳米活性炭吸附丝裂霉素C腹腔化疗的实验研究[J].中华肿瘤杂志，2006，28(4):257-260.
[2]孙岚,张英鸽,杨留中.纳米活性炭在小鼠体内的分布、存留及淋巴靶向性[J].军事医学科学院院刊，2005，29(4):349-351.
[3]张李，梁寒.纳米活性炭的特性及其在胃癌治疗中的应用[J].国际肿瘤学杂志，2007，34（1）：52-55.
[4] Hagiwara A, Torii T, Sawai K, et al. Local injection of anti-cancer drugs bound tocarbon particles for early gastric cancer-a pilot study[J]. Hepatogastroenterology，2000，47(32):575-578.
[5]张英鸽,孙岚,曲秋莲.纳米活性炭的制备及应用，中国专利. ZL03131497.X.
[6]苏伟，周理.高比表面积活性炭制备技术的研究进展[J].化学工程，2005，33(2)：44-47.
[7]孙明磊，温玉明，王昌美，等.淋巴靶向平阳霉素—活性炭纳米微粒的研制[J].华西口腔医学杂志，2004，22(3)：183-185.
[8]董怡民，沓小容，姚崇顺，等.纳米活性炭对氟尿嘧啶的吸附和缓释作用的实验研究[J].中国医科大学学报，2004，33(3)：205-211.
[9]张英鸽，纳米毒理学，中国协和医科大学出版社，2010，第一版：264-265.
[10]李晓林，张艳芳，唐凯，等.纳米二氧化钛颗粒体外对人肺腺癌A549细胞的毒性效应[J].第二军医大学学报，2011，32（10）：1091-1095.
[11]吴秋云，唐萌，谢彦昕，等.不同粒径纳米二氧化硅的体外细胞膜毒性作用[J].中国生物医学工程学报，2010，29（3）：437-445.
[12]杨红，吴秋云，唐萌，等.纳米氧化硅的体外细胞毒性[J].东南大学学报(自然科学版)，2008，38（6）：1054-1060.镍对体外培养内皮细胞的影响[J].现代生物医学进展，2010，10（2）：224-228.
[1]李章芳，沈洁。二甲双胍对肿瘤抑制作用的研究进展[J].中华临床医师杂志，2011，5（17:5081-5083.
[2] Vazquez-Martin A, Oliveras-Ferraros C, Cufi S, Del Barco S, Martin-Castillo B,Menendez JA. Metformin regulates breast cancer stem cell ontogeny bytranscriptional regulation of the epithelial-mesenchymal transition (EMT) status[J].Cell Cycle2010，9:3807-14.
[3] Vazquez-Martin A, Oliveras-Ferraros C, Cu fi S, Del Barco S, Martin-Ca stillo B,Menendez JA. Metformin regulates breast cancer stem cell ontogeny bytranscrip-tional regulation of the epithelial–mesenchymal transition (EMT) status[J].Cell Cycle2010，9:3807-3814.
[4] Hirsch HA, Iliopoulos D, Tsichlis PN, Struhl K. Metformin selectively targetscancer stem cells，and acts together with chemotherapy to block tumor growth andpro-long remission[J]. Cancer Res2009，69：7507-11.
[5] Iliopoulos D, Hirsch HA, Struhl K. Metformin decreases the dose ofchemotherapy for prolonging tumor remission in mouse xenografts involving multiplecancer cell types[J]. Cancer Res2011，71:3196-201.
[6]国家药典委员会编1中华人民共和国药典(二部)[S].2005年版.北京:化学工业出版社，2005：457、附录36.
[7] Aginara A, Takahashi T, AanaiK, et al. Selective drug delivery to peritum oralregion lymphatics by local injection of aclarubicin adsorbed on activated carbonparticles in patients with breast cancer pilot study[J]. Anticancer Drug，1997，8(7):666-670.
[8] Wang Guijuan, Cao Mingli, Yu Yongfu. Study on preparation of Al-pillaredMontmorillonite and its Adsorption[J]. Industrial Minerals and Processing，2003，11:9-11.
[9] Yoshizawa T, Yamashita A, Luo Y. Fumonisin occurrence in corn from high andlow risk areas for human esophageal cancer in China [J]. Appl Environ Microbiol,1994，60(5):1626-1629.
[10] Yang Zhong, Ma Shuzheng and Zhang Yingge. Using activated carbonnanoparticles to decrease the genotoxicity and teratogenicity of anticancertherapeutic agents[J]. J Nanosci Nanotechnol.2010，10:1-7.
[11] SINGHR, Jr LILLARD JW. Nanoparticle-based targeted drug delivery[J]. ExpMol Pathol2009,86(3):215-223.
[12] TA H T, DASS C R, DUNSTAN D E. Injectable chitosan hydrogels for localisedcancer therapy[J]. J Control Release，2008，126(3):205-216.
[13] TRAN M A, WATTS R J, ROBERTSON G P. Use of liposomes as drug deliveryvehicles for treatment of melanoma[J]. Pigm Cell Melanoma R，2009，22(4):388-399.
[14] JABR-MILANE L S, van VLERKEN L E, YADAV S, et al. Multi-functionalnanocarriers to overcome tumor drug resistance [J]. Cancer Treat Rev，2008，34(7):592-602.
[15] Yih TC, Al-Fandi M. Engineered nanoparticles as precise drug delivery systems.J Cell Biochem2006，97:1184-90.
[16] Suphiya Parveen, MS, Ranjita Misra, MS, Sanjeeb K. Sahoo, PhD. Nanoparticles:a boon to drug delivery, therapeutics, diagnostics and imaging[J].Nanomedicine:Nanotechnology, Biology, and Medicine8(2012):147–166.
[1] Gupta PB, Onder TT, Jiang G, et al. Identification of selective inhibitors of cancerstem cells by high-throughput screening. Cell,138:645–659.
[2]Sharon R. Pinea, Blair Marshallb et al. Lung cancer stem cells. Disease Markers24:257–266.
[3]Yin, S., Li, J., Hu, C. et al. CD133positive hepatocellular carcinoma cells possesshigh capacity for tumorigenicity[J]. Int J Cancer.2007,120(7):1444-1450.
[4] Ma, S., Chan, K. W., Hu, L. et al. Identification and characterization oftumorigenic liver cancer stem/progenitor cells[J]. Gastroenterology.2007,132(7):2542-2556.
[5] Yang, W., Yan, H. X., Chen, L. et al. Wnt/beta-catenin signaling contributes toactivation of normal and tumorigenic liver progenitor cells[J]. Cancer Res.2008,68(11):4287-4295.
[6] Yamashita, T., Ji, J., Budhu, A. et al. EpCAM-positive hepatocellular carcinomacells are tumor-initiating cells with stem/progenitor cell features[J]. Gastroenterology.2009,136(3):1012-1024
[7] Wu, A., Wiesner, S., Xiao, J. et al. Expression of MHC I and NK ligands onhuman CD133+glioma cells: possible targets of immunotherapy[J]. J Neurooncol.2007,83(2):121-131
[8] Song, W., Li, H., Tao, K. et al. Expression and clinical significance of the stem cellmarker CD133in hepatocellular carcinoma[J]. Int J Clin Pract.2008,62(8):1212-1218
[9]Horst, D., Scheel, S. K., Liebmann, S. et al. The cancer stem cell marker CD133has high prognostic impact but unknown functional relevance for the metastasis ofhuman colon cancer[J]. J Pathol.2009,219(4):427-434
[10]Yang L, Parkin DM, Li L, et al. Time trends in cancer mortality in China:1987－1999[J]. Int J Cancer,2003,106(5):771－783.
[11] de Villa V, Lo CM. Liver transplantation for hepatocellular carcinoma inAsia[J]. Oncologist,2007,12(11):1321－1331.
[12]孙力超，赵璇，孙立新，等，肝癌干细胞抗体靶向治疗的实验，肿瘤防治研究，2011，38（6）：609-614
[13] Yang ZF, Ho DW, Ng MN, et al. Significance of CD90+cancer stem cells inhuman liver cancer[J]. Cancer Cell,2008,13(2):153－166.
[14] Al－Hajj M, Wicha MS, BenitoHernandez A, et al. Prospective identificationof tumorigenic breast cancer cells[J]. Proc Natl Acad Sci USA,2003,100(7):3983－3988.
[15]Haraguchi N, Ohkuma M, Sakashita H, et al. CD133+CD44+populationefficiently enriches colon cancer initiating cells[J]. Ann Surg Oncol,2008,15(10):2927－2933.
[16]Tirino V, Camerlingo R, Franco R, et al. The role of CD133in theidentification and characterisation of tumourinitiating cells in nonsmallcell lungcancer [J]. Eur J Cardiothorac Surg,2009,36(3):446－453.
[17]Taylor RA, Toivanen R, Risbridger GP. Stem cells in prostate cancer: treatingthe root of the problem[J]. Endocr Relat Cancer,2010,17(4): R273－R285.
[18] Ma S, Chan KW, Hu L, et al. Identification and characterization oftumorigenic liver cancer stem/progenitor cells[J]. Gastroenterology,2007,132(7):2542－2556.
[19]梁晨,单娟娟,沈俊杰.肝癌细胞系Huh7中肿瘤干细胞分离及鉴定,浙江理工大学学报,2001,28(4):602-605.
[20]Dontu G, Abdallah WM, Foley JM, et al. In vitro propagation andtranscriptional profiling of human mammary stem/progenitor cells[J]. Genes Dev,2003,17(10):1253－1270.
[21]Toma J G, Akhavan M, Fernandes KJ, et al. Isolation of multipotent adultstem cells from the dermis of mammalian skin[J]. Nat Cell Biol,2001,3(9):778－784.
[22]Yin AH, Miraglia S, Zanjani ED, et al AC133a novel marker for humanhematopoietic stem and progenitor cells[J]. Blood,1997,90:5002-5012
[23]Singh SK, Clarke D. Identification of a cancer stem cell in human braintumors[J]. Cancer Res,2003,63:58212-58218.
[24]Hadnagy A，Gaboury L，Beanlieu R，et al. SP an alysis may be used to identifycancer stem cell populations. Exp Cell Res2006;312(19):3701-3710.
[25] Ma S，Lee TK，Zheng B-J，et al. CD133+HCC cancer stem cells conferchemoresistance by preferential expression of the Akt/PKB survival pathway.Oncogene2008;27(12):1749-1758.
[1] AL-HAJJM, CLARKEMF. Self-renewal and solid tumor stem cells[J]. Oncogene,2004,23(43):7274-728.
[2] Hadnagy A，Gaboury L，Beanlieu R，et al. SP an alysis may be used to identifycancer stem cell populations[J]. Exp Cell Res2006,312(19):3701-3710.
[3] Ma S，Lee TK，Zheng B-J，et al. CD133+HCC cancer stem cells conferchemoresistance by preferential expression of the Akt/PKB survival pathway[J].Oncogene2008,27(12):1749-1758.
[4] Hirsch HA, Iliopoulos D, Tsichlis PN, et al. Metformin selectively targets cancerstem cells, and acts together with chemotherapy to block tumor growth and prolongremission[J]. Cancer Res,2009,69:7507–7511.
[5] Shank JJ, Yang K, Ghannam J,et al., Metformin targets ovarian cancer stem cellsin vitro and in vivo[J]. Gynecol Oncol,2012, DOI/10.1016/j.ygyno.2012.07.115.
[6] Alejandro V M, Cristina OF,Sonia D B, et al. The anti-diabetic drug metforminsuppresses self-renewal and proliferation of trastuzumab-resistant tumor-initiatingbreast cancer stem cells[J]. Breast Cancer Res Treat,2011,126:355–364.DOI10.1007/s10549-010-0924-x.
[7] ChangXiao Liu. Research and application of targeting nano-drugs[J]. NanoBiomed.Eng,2011,3(2),73-83.
[8] Grimshaw MJ, Cooper L, Papazisis K, et al. Mammosphere culture of metastaticbreast cancer cells enriches for tumorigenic breast cancer cells[J]. Breast Cancer Res,2008,10: R52.
[9] MAEDAH,WUJ, SAWAT, et al.Tumor vascular permeability and the EPR effectin macromolecular therapeutics: a review[J]. J Control Release,2000,65(2):271-284.
[10] A.K. Iyer, G. Kha led, J. Fang, H. Maeda, Exploiting the enhanced permeabilityand retention effect for tumor targeting, Drug Discovery Today11(2006)812–818.
[11] Dontu G, Abdallah WM, Foley JM, et al. In vitro propagation and transcriptionalprofiling of human mammary stem/progenitor cells[J]. Genes Dev2003,17:1253–1270.
[12] Dontu G, Jackson KW, McNicholas E, et al. Role of Notch signaling in cell-fatedetermination of human mammary stem/progenitor cells[J]. Breast Cancer Res,2004,6:605–615.
[13] Marquardt J U, Factor V M, Thorgeirsson S S. Epigenetic regulation of cancerstem cells in liver cancer: Current concepts and clinical implications[J]. Journal ofHepatology,2010,53:568–577.
[14] Li C, Liu VW, Chan DW, Yao KM, Ngan HY. LY294002and metformincooperatively enhance the inhibition of growth and the induction of apoptosis ofovarian cancer cells[J]. Int J Gynecol Cancer2012,22:15-22.
[1] J.D. Byrne, T. Betancourt, L. Brannon-Peppas, Active targeting schemes fornanopartic le systems in cancer therapeutics[J]. Adv. Drug Deliv. Rev.60(2008):1615–1626.
[2] Y.H. Bae, Drug targeting and tumor heterog eneity[J]. J. Control. Release133(2009):2–3.
[3] S.H. Jang, M.G. Wientjes, D. Lu, J.L.S. Au, Drug delivery and transport to solidtumors, Pharmaceutical Research[J].2003(20):1337–1350.
[4] J.R. Less, T.C. Skalak, E.M. Sevick,R.K.Jain, Microvascular architecture in amammary carcinoma: br-anching patterns and vessel dimensions[J]. Cancer Research1991(51):265–273.
[5] F. Yuan, M. Dellian, D. Fukum ura, M. Leunig, D.A. Berk, V.P. Torchilin, R.K.Jain,Vascular-permeability in a human tumor xenograft—molecular-size dependenceand cutoff size[J]. Cancer Research55(1995):3752–3756.
[6] W.G. Roberts, G.E. Palade, Neovasculature induced by vascular endothelial growthfactor is fenestrated[J]. Cancer Research57(1997):765–772.
[7] Yinghuan Li, Jie Wang, M. Guillaume Wientjes, Jessie L.-S. Au. Delivery ofnanomedicines to extracellular and intracellular compartments of a solid tumor[J].Advanced Drug Delivery Reviews64(2012):29–39.
[8]S.H.Jang, M.G.Wientjes, D.Lu,J.L.S. Au, Drug delivery and transport to solidtumors[J]. Pharmaceutical Research20(2003):1337–1350.
[9] W. Peters, M. Teixeira, M. Intaglietta, J.F. Gross, Microcirculatory studies in ratmammary carcin oma. I. Transparent chamber method, development ofmicrovasculature, and pressures in tumor vessels[J]. J. Natl. Cancer Inst.65(1980):631–642.
[10] F. Yuan, M. Dellian, D. Fukum ura, M. Leunig, D.A. Berk, V.P. Torchilin, R.K.Jain,Vascular-pe rmeability in a human tumor xenograft—molecular-size dependenceand cutoff size[J]. Cancer Research55(1995):3752–3756.
[11] C.H. Heldin, K. Rubin, K. Pietras, A. Ostman, High interstitial fluid pressure—an obstacle in cancer therapy[J]. Nat. Rev. Cancer4(2004):806–813.
[12] AL-HAJJM, CLARKEMF. Self-renewal and solid tumor stem cells[J]. Oncogene,2004,23(43):7274-728.
[13] Gupta PB, Onder TT, Jiang G, et al. Identification of selective inhibitors ofcancer stem cells by high-throughput screening[J]. Cell2009:138:645–659.
[14] Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer and cancerstem cells[J]. Nature2001，414:105–111.
[15] Jordan CT, Guzman ML, Noble M. Cancer stem cells[J]. N Engl J Med2006，355:1253–1261.
[16] Sharon R. Pinea, Blair Marshallb and Lyuba Varticovski. Lung cancer stemcells[J]. Disease Markers24(2008)257–266.
[17] Nguyen DX, Bos PD, Massague J. Metastasis: from dissemination toorgan-specific colonization[J]. Nat Rev Cancer2009;9:274–284.
[18] Kim MY, Oskarsson T, Acharyya S, Nguyen DX, Zhang XH, Norton L,et al.Tumor self-seeding by circulating cancer cells[J]. Cell2009;139:1315–1326.
[19] Jordan CT, Guzman ML, Noble M. Cancer stem cells[J]. N Engl J Med2006,355:1253–1261.
[20] Li L, Neaves WB. Normal stem cells and cancer stem cells: the niche matters[J].Cancer Res2006,66:4553–4557.
[21] Bissell MJ, Labarge MA. Context, tissue plasticity, and cancer: are tumor stemcells also regulated by the microenvironment?[J]. Cancer Cell2005,7:17–23.
[22] Polyak K, Weinberg RA. Transitions between epithelial and mesenchymal states:acquisition of malignant and stem cell traits[J]. Nat Rev Cancer2009,9:265–273.
[23] Jaiswal S, Jamieson CH, Pang WW, Park CY, Chao MP, Majeti R, et al. CD47isupregulated on circulating hematopoietic stem cells and leukemia cells to avoidphagocytosis[J]. Cell2009,138:271–285.
[24] Lu J, Fan T, Zhao Q, Zeng W, Zaslavsky E, Chen JJ, et al. Isolation of circulatingepithelial and tumor progenitor cells with an invasive phenotype from breast cancerpatients[J]. Int J Cancer2010,126:669–683.
[25] Ahn JB, Rha SY, Shin SJ, Jeung HC, Kim TS, Zhang X, et al. Circulatingendothelial progenitor cells (EPC) for tumor vasculogenesis in gastric cancerpatients[J]. Cancer Lett2009,288:124–132.
[26] Hermann PC, Huber SL, Herrler T, Aicher A, Ellwart JW, Guba M, et al.Distinctpopulations of cancer stem cells determine tumor growth and metastatic activity inhuman pancreatic cancer[J]. Cell Stem Cell2007,1:313–323.
[27] Aktas B, Tewes M, Fehm T, Hauch S, Kimmig R, Kasimir-Bauer S. Stem celland epithelial-mesenchymal transition markers are frequently overexpres-sed incirculating tumor cells of metastatic breast cancer patients[J]. Breast Cancer Res2009;11:R46.
[28] Liu R, Wang X, Chen GY, Dalerba P, Gurney A, Hoey T, et al. The prognosticrole of a gene signature from tumorigenic breast-cancer cells[J]. N Engl J Med2007;356:217–226.
[29] Yang ZF, Ngai P, Ho DW, Yu WC, Ng MN, Lau CK, et al. Identification of localand circulating cancer stem cells in human liver cancer[J].Hepatology2008,47:919–928.
[30] Clarke MF, Fuller M. Stem cells and cancer: two faces of eve[J]. Cell2006;124:1111–1115.
[31] Huff CA, Matsui W, Smith BD, Jones RJ. The paradox of response and survivalin cancer therapeutics. Blood2006;107(2):431-434.
[32] SINGHR, Jr LILLARD JW. Nanoparticle-based targeted drug delivery[J]. ExpMol Pathol2009,86(3):215-223.
[33] TA H T, DASS C R, DUNSTAN D E. Injectable chitosan hydrogels for localisedcancer therapy[J]. J Control Release,2008,126(3):205-216.
[34] TRAN M A, WATTS R J, ROBERTSON G P. Use of liposomes as drug deliveryvehicles for treatment of melanoma[J]. Pigm Cell Melanoma R,2009,22(4):388-399.
[35] JABR-MILANE L S, van VLERKEN L E, YADAV S, et al. Multi-functionalnanocarriers to overcome tumor drug resistance [J]. Cancer Treat Rev,2008,34(7):592-602.
[36] Yih TC, Al-Fandi M. Engineered nanoparticles as precise drug deliverysystems[J]. J Cell Biochem2006;97:1184-90.
[37] Suphiya Parveen, MS, Ranjita Misra, MS, Sanjeeb K. Sahoo, PhD. Nanoparticles:a boon to drug delivery, therapeutics, diagnostics and imaging[J].Nanomedicine:Nanotechnology, Biology, and Medicine8(2012):147–166.
[38] LEEES, KIMD, YOUNY S, et al. A novel virus-mimetic nanogelvehicle[J].Angew Chem Int Ed,2008,47(13):2418-2421.
[39] LEEES, GAOZ, KIMD, et al. Super pH-sensitivemultifunctional polymericmicelle for tumor pH(e) specific TAT exposure and multidrug resistance: in vivoefficacy[J]. J ControlRelease,2008,129(3):228-236.
[40] KOBAYASHI T, ISHIDAT, OKADAY, et al. Effect of transferrinreceptor-targeted liposomal doxorubicin in P-glycoproteinmediated drug resistanttumor cells[J]. Int J Pharm,2007,329(1):94-102.
[41] MOHAJERG, LEEES, BAEYH. Enhanced intercellular retention activity ofnovel pH-sensitive polymeric micelles in wild and multidrug resistant MCF-7cells[J].PharmRes,2007,24(9):1618-1627.
[42] LEEES, NAK, BAEYH. Doxorubicin loaded pHsensitive polymeric micelles forreversal of resistant MCF-7tumor[J].J ControlRelease,2005,103(2):405-418.
[43] MAEDAH,WUJ, SAWAT, et al Tumor vascular permeability and the EPR effectin macromolecular therapeutics: a review[J]. J Control Release,2000,65(2):271-284.
[44] A.K. Iyer, G. Kha led, J. Fang, H. Maeda, Exploiting the enhanced permeabilityand retention effect for tumor targeting[J]. Drug Discovery Today11(2006)812–818.
[45] SHEKHARC. Lean and mean: nanoparticlebased delivery improves performanceof cancer drugs[J]. Chem Biol,2009,16(4):349-350.
[46] LI SD, HUANGL. Pharmacokinetics and biodistribution of nanoparticles[J].MolPharmaceutics,2008,5(4):496-504.
[47] OGAWARAK, UNK, TANAKAK, et al.In vivo antitumor effect of PEGliposomal doxorubicin(DOX) in DOX-resistant tumor-bearing mice:involvement ofcytotoxic effect on vascular endothelial cells[J]. J Control Release,2009,133(1):4-10
[48] SENGUPTA S, EAVARONED, CAPILA I, et al. Temporal targeting of tumourcells and neovasculature with a nanoscale delivery system[J].Nature,2005,436(7050):568-572.
[49] Lim G. Pelosi, M. Barisella, F. Pasini, M.E. Leon, G. Veronesi, L. Spaggiari, F.Fraggetta, A. Iannucci, M. Masullo, A. Sonzogni, F. Maffini and G. Viale, CD117immunoreactivity in stage I adenocarcinoma and squamous cell carcinoma of the lung:relevance to prognosis in a subset of adenocarcinomapatients [J]. Mod Pathol17(2004),711–721.
[50] M. Gutova, J. Najbauer, A. Gevorgyan, M.Z. Metz, Y.Weng, C.C. Shih and K.S.Aboody, Identification of uPAR-positive Chemoresistant Cells in Small Cell LungCancer[J]. PLoS ONE2(2007), e243.
[51] Zhang L, Yao HJ, Yu Y, Zhang Y, Li RJ, Ju RJ, et al. Mitochondrial targetingliposomes incorporating daunorubicin and quinacrine for treatment of relapsed breastcancer arising from cancer stem cells[J]. Biomaterials2012,33:565-582.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |